Combination of Binary System with Ki67 Immunohistochemical Staining: a Reliable Grading Method for Oral Epithelial Dysplasia

Statement of the Problem: Oral epithelial dysplasia (OED) is a common potentially malignant lesion of oral cavity that should be managed to prevent likely malignant transformation. Purpose: Here, we present a combination of binary grading system with complementary immunohistochemical (IHC) staining for Ki67 biomarker to provide a reproducible OED grading system. Materials and Method: In this cross-sectional study, seventy out of one hundred OED specimens, which were accompanied by IHC stained microscopic slides for Ki67 antigen were evaluated by four independent oral pathologists. Both three-tier and binary grading systems based on WHO microscopic criteria were employed , blindly in a four-step method with at least two-week interval between each observation. Intra- and inter-observational reliability was assessed using Kappa statistical analysis. Results: OED diagnosis based on binary system showed significant intra-observer reliability comparing to three-tier system without biomarker. Moreover, OED diagnosis based on binary system using Ki67 biomarker showed significant inter-observer reliability comparing to diagnosis in three tier system and based on binary system without Ki67 biomarker showed significant inter-observer reliability comparing to diagnosis based on three-tier system without Ki67. Conclusion: Here, we found that application of IHC staining for Ki67 biomarker in binary system might provide a more reliable grading method for oral pathologist form different educational background.


Introduction
Oral cancer is one of the most common cancers in human population with high morbidity and mortality rates [1][2]. Prediction of probability of malignant change in dysplastic oral epithelium, as a potentially malignant lesion is a clinical concern for both pathologists and clinicians, which has a great impact on therapeutic approaches [3][4]. Over the past decades, several grading systems of oral epithelial dysplasia (OED) have been introduced to provide a reliable classification method with sharply defined borders and significant reproducibility [6][7]. Although there is an agreement among investigators that all OEDs do not necessarily transform to an invasive carcinoma, it is inevitable to guide surgeons, facing this potentially malignant lesion, using a clearly interpretable scoring system, which is well-descriptive and fulfills the majority of microscopic features [8][9].
Most well-known grading systems are developed based on microscopic criteria, introduced by World Health Organization (WHO) in 2005 and revised in 2017 [10].
Considering these criteria, OED is scored in a three-graded system (mild, moderate and severe), according to the level of extension of microscopic criteria along epithelial tissue, or in a two-graded system (low-risk, highrisk) according to the severity of epithelial involvement by cellular atypia and architectural changes [11].
Despite all the efforts to propose a grading system with restricted histopathologic subjectivity, emerging of ancillary objective biomarkers may be necessary in routine laboratory practice for microscopic evaluation of the specimens, suspicious of epithelial dysplasia. Over recent years, a wide range of immunohistochemically (IHC) detectable proteins associated with risk of oral cancer initiation is proposed. These proteins are mostly depicted in cancer hallmarks [12]. Ki67, routinely used proliferation marker, is commonly utilized to make definite diagnosis in all types of cancers. Additionally, a recent study has shown that Ki-67 may be helpful in OED stratification by significant nuclear expression, particularly in high-risk dysplasia [13].
Among all OED grading systems and ancillary biomarkers, in the present investigation, we compared two, binary and three-tier WHO grading systems, with and without supplementary IHC study (proliferation marker) to assess their intra-and inter-observer reliabilities.

Case collection
The present study was approved by Ethical Committee  were reviewed by oral pathologists based on both binary system and three-tier WHO classification (Table 2), blindly. The data was recorded for each system, separately.  Step 2: Two weeks after step 1, all H&E stained microscopic slides (100 cases) were reviewed again by oral pathologists based on both binary system and three-tier WHO classification, blindly. The data was recorded again for each system, separately.  Step 3: Four weeks after step 2, all H&E stained with accompanying IHC stained (for Ki67 biomarker) microscopic slides (70 cases) were reviewed by oral pathologists based on both binary system and three-tier WHO classification, blindly. The data was recorded for each system, separately.  Step 4: Two weeks after step 3, all H&E stained with accompanying IHC stained (for Ki67 biomarker) microscopic slides (70 cases) were reviewed by oral pathologists based on both binary system and three-tier WHO classification, blindly. The data was recorded again for each system, separately.

Statistical analysis
The collected data was analyzed using SPSS software 25. Intra-and inter-observer reliability was evaluated using weighted Kappa and statistic values less than 0.05 were considered significant. For multiple comparisons, Bonferroni adjustment was applied on p values [14].

Intra-observer reliability
Statistical analysis of intra-observer reliability demonstrates the below findings (Table 3): -Binary system in combination with Ki67 showed significant intra-observer reliability in comparison with three-tier system without Ki67.  -Twenty-five percent of the cases diagnosed using three -tier system in combination with Ki67 showed significant intra-observer reliability comparing to the cases High-risk: at least four architectural changes and five cytological changes Severe: Criteria for dysplasia observed in greater than two-third of the epithelium diagnosed using three-tier system without Ki67.
-Fifty percent of the cases diagnosed using binary system without Ki67, showed significant intra-observer reliability comparing to the cases diagnosed using three-tier system in combination with Ki67.
Fifty percent of the cases diagnosed using binary system in combination with Ki67 showed significant intra-observer reliability comparing to the cases diagnosed using three-tier system in combination with Ki67.
Twenty-five percent of the cases diagnosed using binosed using binary system without Ki67.
-OED diagnosis based on binary system (with or with-

Inter-observer reliability
Statistical analysis of inter-observer reliability demonstrates the below findings (Table 4): -Two-third of the cases diagnosed using binary system (with/ without Ki67) showed significant inter-observer reliability comparing to the cases diagnosed using threetier system without Ki67.
-Two-third of the cases diagnosed using binary system without Ki67 showed significant inter-observer reliability comparing to the cases diagnosed using three-tier system without Ki67.
-One-third of the cases diagnosed using three-tier system in combination with Ki67 showed significant interobserver reliability comparing to the cases diagnosed using three-tier system without Ki67.
-No significant inter-observer reliability was found betw-een three-tier system in combination with Ki67 comparing with diagnosis and binary system in combination with Ki67.
-No significant inter-observer reliability was found between binary system in combination with Ki67 comparing to binary system without Ki67.
-OED diagnosis based on binary system using Ki67 biomarker showed significant inter-observer reliability comparing to diagnosis in three tier system (with or without Ki67). Moreover, diagnosis based on binary system without Ki67 biomarker showed significant inter-observer reliability comparing to diagnosis based on three-tier system without Ki67 (Table not shown).

Discussion
Oral squamous cell carcinoma commonly arises from OED, which necessitates therapeutic management of these potentially malignant lesions as a serious clinical challenge [15]. A recently published study has confirmed that binary grading system is more reproducible and also more reliable prognostic scale comparing to widely used three-tier system [16]. Additionally, it seems clinicians can make definite decision to manage OED, especially more challenging cases with moderate dysplasia, with confidence using the binary system [9]. Our study showed similar results that excluding ancillary biomarker, binary system has both intra-and inter-observer reliability in comparison with three-tier system. There are also several intervening factors with negative impact on making an accurate diagnosis in OED.
The most important factor is inflammation in adjacent connective tissue, which induces cellular atypia mimicking true dysplastic changes [20]. In our study, all observers are recommended to consider inductive cellular atypia in cases with inflammation in superficial connective tissue.
It should additionally be mentioned that prognostic efficacy of the proposed grading method as an indicator of carcinomatous transformation is not evaluated in the present study which compel us for further investigation.

Conclusion
In this study, we proposed a combination of binary grading system, based on WHO microscopic criteria, using both H&E and IHC stained for Ki67 protein microscopic slides. This method can simply improve objectivity of the binary system, which is well accepted by the majority of surgeons, and confine intra-/interobserver discrepancies among pathologists from different educational background with unlike professional experiences.

Acknowledgments
This study was D.D.S. thesis of Negar Akafzadeh and financially supported by School of Dentistry, Tehran University of Medical Sciences (Grant # 9211272032).
We would like to thank Dr. Narges Hajiani (for her kind support to collect the formalin-fixed, paraffin-embedded specimens from laboratory archive) and Fatemeh Falahati Dowlatabadi (for her valuable support to provide high quality microscopic slides).